Molecular Analysis of DNA Repair Defect and Nnurological Abnormalities in Group a Xeroderma Pigmentosum

点击翻译按钮获取中文摘要

We cloned a mouse DNA excision repair gene which complements the defect of group A xeroderma pigmentosum (XP) and named it the XPAC gene. We also cloned a human XPAC cDNA that encodes 273 amino acid residues and a human XPAC gene that is about 25kb long and is split into six exons. Expression of XPAC cDNA conferred UV-resistance on several group A XP cell lines, but on lines of other XP groups. Almost all group A XP lines tested showed abnormality or absence of XPAC mRNA. These results indicate that a defective XPAC gene causes group A XP. Human XPAC cDNA encodes a hydrophilic protein of relative molecular mass 31K. The XPC protein contains C4 type zinc-finger motif, indicating that it interacts directly with DNA. The recombinant fused XPAC protein was produced in E. coli by T7 expression system and microinjection of the recombinant xpac protein restored UV or 4NQO-induced unscheduled DNA synthesis in group A XP cells. The polylonal anti xpac protein antibody was elicited by injecting … More the recombinant xpac protein into rabbit. Immunoprecipitation of crude cell extract with this antibody and SDS-PAGE detected about 40K and 38K xpac protein in normal human cells, but not in group A XP cells. Immunofluorescence study revealed that xpac protein localized in the cell nucleus.The molecular basis of group A XP was then investigated. We found three different kinds of mutations of XPAC gene in Japanese group A XP patients. One was a G -> C transversion at the 3' splice acceptor site of intron 3, which altered the obligatory AG acceptor dinucleotides to AC, creating two abnormally spliced mRNA forms. This single base substitution creates a new cleavage site for AlwNI restriction endonuclease. Analysis of AlwNI RFLP showed a high frequency of this mutation in Japanese group A XP patients. Second one was C -> T transition in exon 6 altering Arg codon (CGA) to a nonsense codon (TGA). One of the Japanese group A XP patients who showed mild skin symptoms with no skin tumors or neurological abnormalities was a homozygote for this mutation. The third one was a T -> A transversion in exon 3 altering Tyr codon (TAT) to a nonsense codon (TAA). Of 21 unrelated Japanese group AXP patients, 2 with severe clinical symptoms had this mutant alleles. Less